Biology subjects

Carnesecchi, J.

Publications and source records attributed to Carnesecchi, J..

6 recordsLinked to original sources

bioRxiv2025

Conserved architecture of a functional lncRNA-protein interaction in the DNA damage response pathway

LncRNAs are typically poorly conserved at the sequence level, which complicates the use of sequence-based approaches to identify conserved functions. The stress-associated lincRNA-p21 is a paradigm for a sequence-divergent lncRNA. In mice, the interaction between lincRNA-p21 and the hnRNP K protein activates the Cdkn1a gene in cis, encoding the cell-cycle regulator p21, a key effector of the cellular stress response to DNA damage. However, the molecular details of this interaction, and its conservation in other species, remained unknown. To address this, we applied an integrated structural and functional approach, combining in silico, in vitro, ex cellulo, and in cellulo assays. We identified a structural element in mouse and human lincRNA-p21 containing a tetranucleotide motif conserved across more than 100 mammals. This element directly mediates weak binding to the hnRNP K protein in both species, which may enable rapid and reversible modulation of Cdkn1a and timely resolution of the DNA damage response. Conserved hnRNP K binding in humans suggests that the mechanism of Cdkn1a activation may also occur in this species. Our findings establish a shared RNA-protein contact between two syntenic but sequence-divergent transcripts, offering a general strategy for identifying functional RNA elements in other lncRNAs with limited sequence conservation.

molecular biology↗

bioRxiv2024

A nanobody-based approach to capture and visualize interactions of binary protein complexes in living cells

Protein interaction networks (or interactomes) are formed progressively, each interaction influencing the next one. Accordingly, a same protein will establish different interactomes depending on its first associated cofactor, thereby diversifying its function in the cell. In contrast to their central role, few methods exist to capture interactomes of dimeric protein complexes. Here, we tackle this issue by introducing an innovative method based on bimolecular fluorescence complementation and the specific binding of a nanobody fused to a proximity-dependent biotinylating enzyme. This method was applied to visualize and capture specific interactions of the cytoplasmic TAZ/14-3-3e and nuclear TAZ/TEAD2 complexes, which are major downstream effectors of the Hippo signaling pathway. Among other interactions, we identified SERPINB4 as a novel regulator of TAZ and 14-3-3e proliferative activity in mesenchymal stromal cells. Molecular dissections in living cells revealed the central role of a unique residue of TAZ for recruiting SERPINB4 specifically in the presence of 14-3-3e. Overall, our work demonstrates the importance of considering binary protein complexes for deciphering interactomes and establishes a novel sensitive method in this perspective.

molecular biology↗

bioRxiv2024

Synergistic DNA and RNA binding of the Hox transcription factor Ultrabithorax coordinates splicing and shapes in vivo homeotic functions

The dual interaction of many transcription factors (TFs) with both DNA and RNA is an underexplored issue that could fundamentally reshape our understanding of gene regulation. We address this central issue by investigating the RNA binding activity of the Drosophila Hox TF Ultrabithorax (Ubx) in alternative splicing and morphogenesis. Relying on molecular and genetic interactions, we uncover a homodimerization-dependent mechanism by which Ubx regulates splicing. Notably, this mechanism enables the decoupling of Ubx-DNA and -RNA binding activity in splicing. We identify a critical residue for Ubx-RNA binding and demonstrate the essential role of Ubx-RNA binding ability for its homeotic functions. Overall, we uncover a unique mechanism for Ubx-mediated splicing and underscore the critical contribution of synergistic DNA/RNA binding for its morphogenetic functions. These findings advance our understanding of co-transcriptional regulation and highlight the significance of TF-DNA/RNA synergistic function in shaping gene regulatory networks in living organisms. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/612310v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@6b3f84org.highwire.dtl.DTLVardef@116f353org.highwire.dtl.DTLVardef@1c0ceb4org.highwire.dtl.DTLVardef@142906c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIUbx homodimerization enables decoupling of DNA- and RNA-dependent splicing regulation C_LIO_LIThe homeodomain K58 amino acid is critical for Ubx-RNA binding ability C_LIO_LIUbx-RNA binding ability is essential for splicing regulation C_LIO_LIDual DNA/RNA binding activities shape Ubx homeotic functions C_LI

molecular biology↗

bioRxiv2021

The Hox transcription factor Ultrabithorax binds RNA and regulates co-transcriptional splicing through an interplay with RNA polymerase II

Transcription Factors (TFs) play a pivotal role in cell fate decision by coordinating distinct gene expression programs. Although most TFs act at the DNA regulatory layer, few TFs can bind RNA and modulate mRNA splicing. Yet, the mechanistic cues underlying TFs function in splicing remain elusive. Focusing on the Drosophila Hox TF Ultrabithorax (Ubx), our work shed light on a novel layer of Ubx function at the RNA level. Transcriptome and genome-wide binding profiles in embryonic mesoderm and Drosophila cells indicate that Ubx regulates mRNA expression and splicing to promote distinct functions in defined cellular contexts. Ubx modulates splicing via its DNA-binding domain, the Homeodomain (HD). Our results demonstrate a new RNA-binding ability of Ubx in cells and in vitro. Notably, the N51 amino acid of the HD, which mediates Ubx-DNA interaction, is non-essential for Ubx-RNA interaction in vitro but is required in vivo. We find that the N51 amino acid is necessary to mediate interaction between Ubx and the active form of the RNA Polymerase II (Pol II S2Phos) in Drosophila cells. By combining molecular and imaging approaches, our results reveal that Ubx mediates elongation-coupled splicing via a dynamic interplay with active Pol II and chromatin binding. Overall, our work uncovered a novel role of the Hox TFs at the mRNA regulatory layer. This could be an essential function for other classes of TFs to control cell diversity.

molecular biology↗

bioRxiv2019

Role of a versatile peptide motif in controlling Hox nuclear export and autophagy in the Drosophila fat body

Hox proteins are major regulators of embryonic development, acting in the nucleus to regulate the expression of their numerous downstream target genes. By analyzing deleted forms of the Drosophila Hox protein Ultrabithorax (Ubx), we revealed the presence of an unconventional Nuclear Export Signal (NES) that overlaps with the highly conserved hexapeptide (HX) motif. This short linear motif was originally described as mediating the interaction with the PBC proteins, a generic and crucial class of Hox transcriptional cofactors in development and cancer. Here we show that the HX motif is involved in the interaction with the major CRM1/Embargoed exportin protein. This novel role was found in several Drosophila and human Hox proteins. We provide evidence that HX-dependent Hox nuclear export is tightly regulated in the Drosophila fat body to control the onset of autophagy. Our results underline the high molecular versatility of a unique short peptide motif for controlling context-dependent activity of Hox proteins at both transcriptional and non-transcriptional levels.

developmental biology↗

bioRxiv2018

The Hox Transcription Factor Ubx stabilizes Lineage Commitment by Suppressing Cellular Plasticity

During development cells become gradually restricted in their differentiation potential by the repression of alternative cell fates. While we know that the Polycomb complex plays a crucial role in this process, it still remains unclear how alternative fate genes are specifically targeted for silencing in different cell lineages. We address this question by studying Ultrabithorax (Ubx), a multi-lineage transcription factor (TF) of the Hox class, in the mesodermal and neuronal lineages using sorted nuclei of Drosophila embryos and by interfering with Ubx in mesodermal cells that have already initiated differentiation. We find that Ubx is a key regulator of lineage development, as its mesoderm-specific depletion leads to the de-repression of many genes normally expressed in other lineages. Ubx silences expression of alternative fate genes by interacting with and retaining the Polycomb Group (PcG) protein Pleiohomeotic (Pho) at Ubx targeted genomic regions, thereby setting repressive chromatin marks in a lineage-dependent manner. In sum, our study demonstrates that Ubx stabilizes lineage choice by suppressing the multi-potency encoded in the genome in a lineage-specific manner via its interaction with Pho. This mechanism may explain why the Hox code is maintained throughout the lifecycle, since it seems to set a block to transdifferentiation in many adult cells.

developmental biology↗